Fusion protein comprising anti-c-met antibody and vegf-binding fragment

ABSTRACT

There are provided a fusion protein formed by coupling of anti-c-Met antibody and VEGF-binding fragment, a bispecific antibody comprising the fusion protein, a polynucleotide encoding the fusion protein, a transformant comprising the polynucleotide, a pharmaceutical composition comprising the bispecific antibody as an active ingredient, and a method for preparing the bispecific antibody which is targeted at c-Met and VEGF at the same time, with improved anticancer and anti-angiogenesis effects, comprising coupling an anti-c-Met antibody with a VEGF-binding fragment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefits of Korean Patent Application No. 10-2013-0033872, filed on Mar. 28, 2013, in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 160,391 Byte ASCII (Text) file named “715587_ST25.TXT,” created on Mar. 27, 2014.

BACKGROUND OF THE INVENTION

1. Field

Provided are a fusion protein comprising an anti-c-Met antibody and a VEGF-binding fragment which are coupled to each other, a bispecific antibody comprising the fusion protein, a polynucleotide encoding the fusion protein, a transformant comprising the polynucleotide, a pharmaceutical composition comprising the bispecific antibody as an active ingredient, and a method for preparing the bispecific antibody targeting c-Met and VEGF at the same time and has improved anticancer and anti-angiogenesis effects, comprising coupling an anti-c-Met antibody with a VEGF-binding fragment.

2. Description of the Related Art

c-Met is a receptor tyrosine kinase (RTK) present at the surface of cells, and binds to its ligand, hepatocyte growth factor (HGF), to promote intracellular signal transduction, thereby promoting the growth of cells. In addition, c-Met is over-expressed in cancer cells, and thereby widely implicated in cancer incidence, cancer metastasis, cancer cell migration, cancer cell invasion, angiogenesis, etc. c-Met is over-expressed in many kinds of cancers and, in particular, most of the patients with over-expressed c-Met tend to have poor prognosis.

Vascular endothelial cell growth factor (VEGF) is also present in normal cells and, in particular, it is secreted from cancer cells and binds to its receptor, VEGF Receptor (VEGFR) to induce angiogenesis, whereby cancer cells are supplied with nutrients necessary for their growth through newly induced blood vessels.

Therefore, both of c-Met and VEGF are of great importance as targets in developing anticancer drugs. There remains a need for development of bispecific antibodies targeting c-Met and VEGF at the same time and therapeutic technologies using the same.

BRIEF SUMMARY OF THE INVENTION

One embodiment provides a fusion protein comprising (a) an anti-c-Met antibody or an antigen-binding fragment of the anti-c-Met antibody and (b) a VEGF-binding fragment.

Another embodiment provides a bispecific antibody targeting c-Met and VEGF, comprising the fusion protein.

Another embodiment provides a polynucleotide encoding the fusion protein.

Another embodiment provides a transformant comprising the polynucleotide.

Another embodiment provides a pharmaceutical composition comprising the bispecific antibody as an active ingredient.

Still another embodiment provides a method of preparing a bispecific antibody targeting c-Met and VEGF and having improved anticancer and anti-angiogenesis effects, comprising coupling an anti-c-Met antibody with a VEGF-binding fragment.

Also provided is a method of preventing or treating a disease induced by c-Met or VEGF comprising administering a pharmaceutical amount of the bispecific antibody to a patient in need thereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic view showing one embodiment of a bispecific antibody.

FIG. 2A is a graph showing the c-Met degradation activities of the bispecific antibodies prepared in one example. The value of a control group (media) treated with no anti-c-Met antibody was set to 100%. The values obtained after treatment with the anti-c-Met antibodies were calculated as relative values against the control group.

FIG. 2B is a graph showing the Akt phosphorylation inhibitory degrees of the bispecific antibodies prepared in one example. The value of a control group (5D5) was set to 100%.

FIG. 2C is a graph showing the MKN45 cell line growth inhibitory degree of the bispecific antibody prepared in one example.

FIG. 2D is a graph showing the NCI-H441 cell line growth inhibitory degrees of the bispecific antibodies prepared in one example.

FIG. 2E is a graph showing the HUVEC proliferation inhibitory degree of the bispecific antibody prepared in one example when HGF is added.

FIG. 3A is a graph showing HUVEC proliferation inhibitory degree of the bispecific antibody prepared in one example when VEGF is added.

FIG. 3B is a graph showing the HUVEC proliferation inhibitory degree of the bispecific antibody prepared in one example when VEGF+HGF are added.

FIG. 3C is a graph showing relative invasion (upper portion) and contains photographs (lower portion).

FIG. 3D is a graph showing the HUVEC invasion inhibitory degree of the bispecific antibody prepared in one example when VEGF+HGF are added. The upper portion is a graph showing the relative fluorescence image areas of each treatment group. The lower portion contains photographs illustrating fluorescence images with each of the identified antibodies

FIG. 3E is a graph showing the cell migration inhibitory degree of the bispecific antibody prepared in one example.

FIG. 4A illustrates the in vivo anticancer effects of the bispecific antibody prepared in one example against U87-MG brain cancer cells. The upper portion of FIG. 4A illustrates tumor volumes according to time. The lower portion of FIG. 4A illustrates the weights of the tumors that were extracted after completion of the experiment

FIG. 4B illustrates the in vivo anticancer effects of the bispecific antibody prepared in one example against NCI-H441 lung cancer cells. The upper portion of FIG. 4B is a graph illustrating tumor volumes according to time. The lower portion is a graph illustrating the weights of tumors that were extracted after completion of treatment.

FIG. 5 is a graph showing the comparison of the c-Met degradation activities of various c-Met antibodies fused with VIG2 according to one example with that of c-Met antibody with no VIG2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can effectively inhibit the growth of cancer cells and angiogenesis by providing a bispecific antibody capable of binding to c-Met and VEGF at the same time, where the bispecific antibody targets c-Met and VEGF at the same time, thereby leading to improved anticancer effects and/or angiogenesis inhibitory effects.

The bispecific antibody capable of binding to c-Met and VEGF at the same time can inhibit the growth itself of cancer cells by targeting c-Met, and at the same time, it can block angiogenesis and prevent the supply of nutrients essential for the growth of cancer cells by targeting VEGF, to inhibit the growth of the cancer cells again, thereby leading to more enhanced synergistic effects in inhibition of cancer cell growth and/or inhibition of angiogenesis.

One embodiment provides a fusion protein comprising an anti-c-Met antibody and a VEGF-binding fragment. The anti-c-Met antibody may include an antigen-binding fragment of an anti-c-Met antibody and a modified form of the antibody, as well as an anti-c-Met antibody. Therefore, the fusion protein may be one formed by coupling of a VEGF-binding fragment, and an anti-c-Met antibody, an antigen-binding fragment of the antibody, or a modified form of the antibody.

In another embodiment, a method of using the fusion protein as a bispecific (dual-targeting) antibody capable of binding to c-Met and VEGF at the same time is provided. In particular, there is provided a bispecific antibody against c-Met and VEGF, comprising a fusion protein formed by coupling of VEGF-binding fragment, and anti-c-Met antibody, an antigen-binding fragment of the antibody, or a modified form of the antibody.

In still another embodiment, there are provided a polynucleotide encoding the fusion protein and a transformant transduced with the polynucleotide. The transformant can be used for the production of the fusion protein or the bispecific antibody.

In still another embodiment, there is provided a pharmaceutical composition comprising the fusion protein or the bispecific antibody as an active ingredient. The pharmaceutical composition may have prevention and/or treatment effects against c-Met and/or VEGF induced diseases. For example, the pharmaceutical composition may be an anticancer drug or an angiogenesis inhibitor.

In still another embodiment, there is provided a method for preventing and/or treating c-Met and/or VEGF induced diseases, comprising administering a therapeutically effective amount of the fusion protein or the bispecific antibody.

In still another embodiment, there is provided a method for preparing a bispecific antibody targeting c-Met and VEGF at the same time and having improved anticancer and anti-angiogenesis effects, comprising culturing the transformant to express the fusion protein, or coupling an anti-c-Met antibody, an antigen-binding fragment of the antibody, or a modified form of the antibody with a VEGF-binding fragment.

In still another embodiment, there is provided a method for preparing c-Met antibody with improved anticancer effects and/or angiogenesis inhibitory effects, by coupling an anti-c-Met antibody, an antigen-binding fragment of the antibody, or a modified form of the antibody with a VEGF-binding fragment.

In still another embodiment, there is provided a composition for detection of c-Met and/or VEGF, comprising the fusion protein as an active ingredient.

In still another embodiment, there is provided a composition for diagnosis of c-Met and/or VEGF induced diseases, comprising the fusion protein as an active ingredient.

VEGF is present in normal cells and particularly, it is secreted from cancer cells and binds to its receptor (VEGFR: VEGF Receptor) to induce angiogenesis, and cancer cells are supplied with nutrients necessary for their growth through newly induced blood vessels. The over-expression of VEGF becomes a cause of various diseases and it is involved with not only cancer incidence but also its bad prognosis such as invasion, metastasis, and so on. For such reasons, VEGF has become a key target in anticancer therapy.

VEGF may originate from mammals including primates such as humans and monkeys, and rodents such as mice and rats. For example, the VEGF proteins may be a polypeptide encoded by a nucleotide sequence (mRNA) provided by GenBank Accession Number NM_(—)001025366.2, NM_(—)001025367.2, NM_(—)001025368.2, NM_(—)001025369.2, NM_(—)001025370.2, NM_(—)001033756.2, NM_(—)001171622.1, NM_(—)001171623.1, NM_(—)001171624.1, NM_(—)001171625.1, NM_(—)001171626.1, NM_(—)001171627.1, NM_(—)001171628.1, NM_(—)001171629.1, NM_(—)001171630.1, NM_(—)001204384.1, NM_(—)001204385.1, NM_(—)003376.5, etc.

The “c-Met” or “c-Met protein” refers to a receptor tyrosine kinase (RTK), which binds hepatocyte growth factor (HGF). The c-Met protein may originate from any kinds of species, and for example, it may originate from primates, such as human c-Met (e.g., NP_(—)000236) or monkey c-Met (e.g., Macaca mulatta, NP_(—)001162100), or rodents such as mouse c-Met (e.g., NP_(—)032617.2) or rat c-Met (e.g., NP_(—)113705.1). For example, c-Met proteins may include a polypeptide encoded by the nucleotide sequence identified as GenBank Accession Number NM_(—)000245, or a polypeptide including the amino acid sequence identified as GenBank Accession Number NP_(—)000236, or extracellular domains thereof. The receptor tyrosine kinase c-Met is implicated in several mechanisms, for example, cancer incidence, cancer metastasis, cancer cell migration, cancer cell invasion, angiogenesis, etc.

The anti-c-Met antibody is a protein specifically recognizing c-Met, and may be a single-targeting antibody recognizing c-Met only. A detailed explanation thereof is as follows.

The antigen-binding fragment of the anti-c-Met antibody may be selected from the group consisting of complementarity determining region (CDR), a combination of CDR and Fc region, scFv, (scFv)₂, Fab, Fab′, or F(ab′)₂ of the anti-c-Met antibody, which is described below.

When the anti-c-Met antibody is used, the VEGF-binding fragment may be coupled to the C-terminus of the anti-c-Met antibody either through a suitable peptide linker or without it. Also, when an antigen-binding fragment of the anti-c-Met antibody is used, the VEGF-binding fragment may be coupled to either the N-terminus or the C-terminus of the antigen-binding fragment of the anti-c-Met antibody, and it may be coupled through a suitable peptide linker or without it.

A modified form of the antibody may include isotypes of human and animal antibodies present in nature and/or Fc of any human antibodies (e.g., IgG1, IgG2, and the like) or animal antibodies in which one or more amino acids constituting a hinge portion are modified. Unless mentioned differently herein, the anti-c-Met antibody may be understood to include such modified forms.

The VEGF-binding fragment of the fusion protein may be a molecule that binds to VEGF, for example, a VEGF-binding protein such as a VEGF receptor, anti-VEGF antibody, and the like, or a fragment including a VEGF-binding region of the VEGF-binding protein, for example, an antigen-binding fragment of the anti-VEGF antibody or a VEGF-binding region of the VEGF receptor. For example, the anti-VEGF antibody may be Bevacizumab, and the antigen-binding fragment of the anti-VEGF antibody may be selected from the group consisting of a complementarity determining region (CDR) or variable region thereof, a combination of a CDR or variable region and Fc region thereof, or an scFv, (scFv)₂, Fab, Fab′, or F(ab′)₂ fragment of the anti-VEGF antibody. The VEGF receptor may be, for example, human VEGF receptor 1 (P17948.2; SEQ ID NO: 109), human VEGF receptor 2 (P35968.2), human VEGF Receptor 3 (P35916.3), and the like. Also, the fragment including a VEGF-binding region of the VEGF-binding protein may be second Ig-like domain 2 (VIG2) or a polypeptide including the VIG2 region within the VEGF receptor. For example, the fragment including the VEGF-binding region of human VEGF receptor 1 may be second Ig-like domain 2 (VIG2; e.g., an amino acid sequence (SEQ ID NO: 110) from 129^(th) to 229^(th) positions of the amino acid sequence (SEQ ID NO: 109; P17948.2)), or a polypeptide including 102 to 1338 consecutive amino acids containing the VIG2 (SEQ ID NO: 110) within the amino acid sequence of SEQ ID NO: 109.

Accordingly, in a concrete embodiment, the VEGF-binding fragment may be an anti-VEGF antibody such as Bevacizumab and the like, an antigen-binding fragment of the anti-VEGF antibody selected from the group consisting of complementarity determining region (CDR), a combination of CDR and Fc region, scFv, (scFv)₂, Fab, Fab′, or F(ab′)₂ of the anti-VEGF antibody, a VEGF receptor (e.g., SEQ ID NO: 109), or a VEGF-binding region of the VEGF receptor (e.g., VIG2 of SEQ ID NO: 110, or polypeptide including 102 to 1338 consecutive amino acids including VIG2 of SEQ ID NO: 110 within the amino acid sequence of SEQ ID NO: 109).

In a concrete embodiment, the fusion protein or the bispecific antibody may be formed by coupling a Fc region of an anti-c-Met antibody as described below, an antigen-binding fragment, or a modified form (variant) thereof with a VEGF-binding fragment, e.g., VIG2 (see FIG. 1).

The anti-c-Met antibody, an antigen-binding fragment of the antibody, or a modified form of the antibody, and the VEGF-binding fragment may be coupled through a linker. The linker may be a peptide linker in length of 1 to about 100 amino acids, particularly about 2 to about 50 amino acids, more particularly about 5 to about 30 amino acids. For example, the peptide linker may include one or more amino acids selected from the group consisting of Gly, Asn, Ser, Thr, and Ala, but not be limited thereto. In a specific embodiment, the linker may be expressed as (GGGGS)n (SEQ ID NO: 116), wherein n is the repeat number of (GGGGS) (SEQ ID NO: 116) units, and in consideration of the efficacy of the bispecific antibody, n may be 1 to about 10, particularly 1 to about 5.

The present invention is characterized by enhancing the pharmacological effects of c-Met antibody by coupling the c-Met antibody or a fragment thereof with a VEGF-binding fragment (e.g., VIG2) to manufacture a bispecific antibody.

The anti-c-Met antibody used in the fusion protein or the bispecific antibody may be any anti-c-Met antibody or any antigen-binding fragment thereof. In particular, the anti-c-Met antibody may recognize a specific region of c-Met, for example a specific region within the SEMA domain, as an epitope. The anti-c-Met antibody may be any antibody or antigen-binding fragment that acts on c-Met to induce c-Met intracellular internalization and degradation.

c-Met, a receptor for hepatocyte growth factor (HGF), may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and contains a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorins-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may include the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region including the amino acid sequence of SEQ ID NO: 71, which corresponds to a range from amino acid residues 106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79) of c-Met protein, is a loop region between the second and the third propellers within the epitopes of the SEMA domain. The region acts as an epitope for the specific anti-c-Met antibody of the present invention.

The term “epitope,” as used herein, refers to an antigenic determinant, which is a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region comprising 5 or more contiguous (consecutive or non-consecutive) amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, 5 to 19 consecutive amino acid residues within the amino acid sequence of SEQ ID NO: 71. For example, the epitope may be a polypeptide including 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, wherein the polypeptide essentially includes the amino sequence of SEQ ID NO: 73 (EEPSQ) serving as an essential element for the epitope. For example, the epitope may be a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope including the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third propellers within the SEMA domain of a c-Met protein. The epitope including the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or antigen-binding fragment according to one embodiment most specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope which has 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody may be an antibody or an antigen-binding fragment thereof, which includes:

(a) a heavy chain variable region comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 including the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 including the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid sequence including 8-19 consecutive amino acids comprising amino acid residues from the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and (c) a CDR-H3 including the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 85, or an amino acid sequence including 6-13 consecutive amino acids comprising amino acid residues from the 1^(st) to 6^(th) positions of SEQ ID NO: 85; and (b) a light chain variable region comprising at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 including the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 including the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 86, or an amino acid sequence including 9-17 consecutive amino acids comprising amino acid residues from the 1^(st) to 9^(th) positions of SEQ ID NO: 89.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas I to VI, below:

Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser(SEQ ID NO: 4),  Formula I

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr(SEQ ID NO: 5),  Formula II

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Asp-Asn-Trp-Leu-Xaa₆-Tyr(SEQ ID NO: 6),  Formula III

wherein Xaa₆ is Ser or Thr,

Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn-Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala (SEQ ID NO: 7),  Formula IV

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Tip, Xaa₉ is His or Gln, and Xaa₁₀ is Lys or Asn,

Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃(SEQ ID NO: 8),  Formula V

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser or Pro, and

Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr(SEQ ID NO: 9),  Formula VI

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, or Lys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. The CDR-H2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85.

The CDR-L1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or the antigen-binding fragment may include a heavy variable region including a polypeptide (CDR-H1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26, and a polypeptide (CDR-H3) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85; and a light variable region including a polypeptide (CDR-L1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including an amino acid sequence selected from the group consisting of SEQ ID NOS 12, 13, 14, 15, 16, 37, 86, and 89.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected to humans for the purpose of medical treatment, and thus chimeric antibodies include been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still include variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies have been developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodies is choosing the optimized human antibodies for accepting CDRs of animal-derived antibodies. Antibody databases, analysis of a crystal structure, and technology for molecule modeling are used. However, even when the CDRs of animal-derived antibodies are grafted to the most optimized human antibody framework, amino acids positioned in a framework of the animal-derived CDRs affecting antigen binding are present. Therefore, in many cases, antigen binding affinity is not maintained, and thus application of additional antibody engineering technology for recovering the antigen binding affinity is necessary.

The anti-c-Met antibodies may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies or antigen-binding fragments thereof may be isolated from a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be synthetic or recombinant.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha 1(α1), or alpha 2(α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region V_(H) that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region V_(L) that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region C_(L).

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” and “specifically recognized” are well known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including antigen-binding regions having the ability to specifically bind to the antigen. In one embodiment, the antibody may be an antigen-binding fragment selected from the group consisting of CDR, a combination of CDR and Fc region, scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂.

Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region C_(H1), has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of C_(H1).

The F(ab′)₂ antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment. Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv.

The antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)₂ fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.

The term “hinge region,” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin may be replaced with a human IgG1 hinge or IgG2 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may be modified by the deletion, insertion, addition, or substitution of at least one (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) amino acid residue on the amino acid sequence of the hinge region so that it exhibit enhanced antigen-binding efficiency. For example, the antibody may include a hinge region including the amino acid sequence of SEQ ID NO: 100(U7-HC6), 101(U6-HC7), 102(U3-HC9), 103(U6-HC8), or 104(U8-HC5), or a hinge region including the amino acid sequence of SEQ ID NO: 105 (non-modified human hinge). Preferably, the hinge region has the amino acid sequence of SEQ ID NO: 100 or 101.

The heavy chain constant region and the light chain constant region other than the CDR region, the heavy chain variable region, or the light chain variable region may be the heavy chain constant region and the light chain constant region of any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.).

In one embodiment of the anti-c-Met antibody or antigen-binding fragment, the variable domain of the heavy chain has the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94 and the variable domain of the light chain has the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or 107.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced by the hybridoma cell line deposited with Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (refer to Korean Patent Publication No. 2011-0047698, the entire disclosures of which are incorporated herein by reference). The anti-c-Met antibody may include all the antibodies defined in Korean Patent Publication No. 2011-0047698.

By way of further example, the anti-c-Met antibody or the antibody fragment may include:

a heavy chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1^(st) to 17^(th) positions is a signal peptide), or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), and the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and

a light chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

For example, the anti-c-Met antibody may be selected from the group consisting of:

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 108.

In another embodiment, the anti-c-Met antibody may include a light chain variable region including the amino acid sequence of SEQ ID NO: 106, a variable domain of a light chain including the amino acid sequence of SEQ ID NO: 107, or a light chain including the amino acid sequence of SEQ ID NO: 108.

The polypeptide of SEQ ID NO: 70 is a light chain including human kappa (κ) constant region, and the polypeptide with the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 (corresponding to position 36 of SEQ ID NO: 68 according to kabat numbering) of the polypeptide with the amino acid sequence of SEQ ID NO: 70 with tyrosine. The production yield of the antibodies may be increased by the replacement. The polypeptide with the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 (position 27e according to kabat numbering in the amino acid sequence from amino acid residues 21 to 240 of SEQ ID NO: 68; positioned within CDR-L1) of SEQ ID NO: 108 with tryptophan. By such replacement, antibodies and antibody fragments comprising such sequences exhibit increased activities, such as c-Met biding affinity, c-Met degradation activity, Akt phosphorylation inhibition, and the like.

In a specific embodiment, the fusion protein or the bispecific antibody may be those formed by coupling the Fc portion of the anti-c-Met antibody or the N-terminal or C-terminal of an antigen-binding fragment of the antibody with a VEGF-binding fragment (e.g., VIG2) via a peptide linker or without it (see FIG. 1).

In a specific embodiment, the fusion protein or the bispecific antibody may include (a) a heavy chain including the amino acid sequence of SEQ ID NO: 62, the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64, the amino acid sequence from 18^(th) to 61^(st) positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66, or the amino acid sequence from 18^(th) to 460^(th) positions of SEQ ID NO: 66, and (b) a light chain including the amino acid sequence of SEQ ID NO: 68, the amino acid sequence from 21^(st) to 240^(th) positions of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70, the amino acid sequence from 21^(st) to 240^(th) positions of SEQ ID NO: 70, or the amino acid sequence of SEQ ID NO: 108.

The VEGF-binding fragment (e.g., VIG2 or a polypeptide including VIG2) is confirmed not only to enhance antibody efficacy but also to remarkably reduce side effects (agonism), even when it is coupled to other anti-c-Met antibodies besides the anti-c-Met antibody as mentioned above (see FIG. 5).

In particular, the c-Met antibodies of which the antibody efficacy and agonism can be improved by being coupled with VEGF-binding fragment may be any one selected from all anti-c-Met antibodies other than the above-described anti-c-Met antibody as well as the above-described anti-c-Met antibody. For example the other anti-c-Met antibody may be one including (a) a heavy chain including the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO: 114 and (b) a light chain including the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 115, more particularly, including (a) a heavy chain including a heavy chain including the amino acid sequence of SEQ ID NO: 112 and (b) a light chain including the amino acid sequence of SEQ ID NO: 113, or including (a) a heavy chain including the amino acid sequence of SEQ ID NO: 114 and (b) a light chain including the amino acid sequence of SEQ ID NO: 115.

The fusion protein formed by coupling of the VEGF-binding fragment, and the anti-c-Met antibody or an antigen-binding fragment of the antibody through a linker or without a linker can be used as a precursor for preparing a bispecific antibody which targets c-Met and VEGF at the same time. The pharmaceutical composition may include a pharmaceutically effective amount of the bispecific antibody, together with a pharmaceutically acceptable carrier, diluent, and/or excipient.

The pharmaceutically acceptable carrier included in the pharmaceutical composition may be any commonly used in the formulations of drugs, and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like, but not limited thereto. Besides these ingredients, the pharmaceutical composition may further include one or more selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and a preservative.

The pharmaceutical composition may be administered through oral or parenteral route. Parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the composition for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the composition may be administered using an optional device capable of delivering an active substance to target cells.

The term “the pharmaceutically effective amount” as used herein refers to an amount at which each active ingredient can exert pharmaceutically significant effects.

The pharmaceutical composition may be administered to mammals including primates such as humans and monkeys and rodents such as rats and mice.

The effective amount of the bispecific antibody in the pharmaceutical composition for a single dose may be prescribed in a variety of ways, depending on factors such as formulation methods, administration manners, age of patients, body weight, gender, pathologic conditions, diets, administration time, administration interval, administration route, excretion speed, and reaction sensitivity. For example, the effective amount of the bispecific antibody for a single dose may be in the range of 0.1 to 50 mg/kg, particularly 1 to 20 mg/kg. The effective amount for the single dose may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms. Alternatively, the pharmaceutical composition may be manufactured to be contained in a multiple dosage container.

The pharmaceutical composition may be a solution in an oil or an aqueous medium, a suspension, a syrup, an emulsifying solution form, or it may be formulated into the form of an extract, powders, granules, a tablet or a capsule, and it may further include a dispersing agent or a stabilizing agent for formulation.

In particular, the pharmaceutical composition may be formulated into an immunoliposome since it contains a bispecific antibody. A liposome containing an antibody may be prepared using any methods well known in the pertinent field. The immunoliposome may be a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide-exchange reaction. A chemical drug, such as doxorubicin, may be further included in the liposome.

The pharmaceutical composition described herein can be used for preventing and/or treating diseases induced by c-Met and VEGF, for example, diseases induced by an increase in copy number and/or increased expression (e.g., overexpression) of c-Met and/or over-expression of VEGF, and particularly, a cancer or metastasis of a cancer. The cancer may be those showing over-expression of c-Met and/or VEGF, and they may include, without limitation, one or more selected from the group consisting of solid cancers, such as, squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancers, brain cancer, osteosarcoma, and the like. In a particular embodiment, the cancer may be brain cancer, stomach cancer, kidney cancer, or lung cancer (e.g., small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, etc.). The cancer may include a metastatic cancer, as well as a primary cancer. In another embodiment, the diseases induced by c-Met and VEGF may be gestational diabetes, diabetic retinopathy, macular degeneration (e.g., wet age-related macular degeneration: wet AMD) and so on.

Thus, a method of preventing and/or treating a c-Met and/or VEGF induced disease is provided, wherein the method comprises administering a pharmaceutically effective amount of the fusion protein or the bispecific antibody to a patient in need of the prevention and/or treatment of c-Met and/or VEGF induced diseases. The prevention and/or treatment method may further include the step of identifying a patient in need of the prevention and/or treatment of c-Met and/or VEGF induced diseases, prior to the administration step.

In addition, the anti-c-Met antibody can function independently from Cbl which is a typical RTK negative regulator, and exhibit c-Met degradation activity via a lysosome pathway, not via a proteasome pathway which is mediated by Cbl. Thus, even if it is administered to patients where Cbl does not normally function due to a mutation of Cbl, an insufficient expression amount of Cbl, a mutation of Cbl binding region of c-Met, and the like, c-Met can be suppressed through the LRIG1 mediator which is another negative regulator functioning independently from Cbl. Accordingly, the administration of the bispecific antibody comprising the anti-c-Met antibody may be particularly advantageous to the patients where Cbl does not function normally.

Also provided herein is a polynucleotide encoding a fusion protein comprising an anti-c-Met antibody or antigen-binding fragment thereof and a VEGF-binding fragment, as well as a recombinant vector comprising the polynucleotide and a recombinant cell containing the polynucleotide or vector. All aspects of the c-Met antibody and antibody fragment, and the VEGF-binding fragment, are as described herein with respect to the fusion protein. The nucleic acid, vector, and host cell can be used for any purpose, for instance, a method of preparing the fusion protein by expressing the nucleic acid or vector in the host cell.

The term “vector” used herein refers to a means for expressing a target gene in a host cell. For example, it includes a plasmid vector, a cosmid vector, and a virus vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector and an adeno-associated virus vector. Suitable recombinant vectors may be constructed by manipulating plasmids often used in the art (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, and the like), a phage (for example, λgt4λB, λ-Charon, λΔz1, M13, and the like), or a virus (for example, SV40, and the like), but not be limited thereto.

In the recombinant vector, the polynucleotides may be operatively linked to a promoter. The term “operatively linked” used herein refers to a functional linkage between a nucleotide expression regulating sequence (for example, a promoter sequence) and other nucleotide sequences. Thus, the regulating sequence may regulate the transcription and/or translation of the other nucleotide sequences by being operatively linked.

The recombinant vector may be constructed for either cloning or expression in a conventional manner. The expression vector may be any ordinary vectors known in the pertinent art for expressing an exogenous protein in plants, animals, or microorganisms. The recombinant vector may be constructed using various methods known in the art.

The recombinant vector may be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when a prokaryotic cell is used as a host cell, the expression vector used generally includes a strong promoter capable of initiating transcription (for example, pL^(λ) promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, and the like), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. When a eukaryotic cell is used as a host cell, the vector used generally includes the origin of replication acting in the eukaryotic cell, for example, a fl replication origin, a SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, or a BBV replication origin, but is not limited thereto. A promoter in an expression vector for a eukaryotic host cell may be a promoter derived from the genomes of mammalian cells (for example, a metallothionein promoter, and the like) or a promoter derived from mammalian viruses (for example, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a cytomegalovirus promoter, a tk promoter of HSV, and the like). A transcription termination sequence in an expression vector for a eukaryotic host cell may be, in general, a polyadenylation sequence.

The recombinant cell may be obtained by transfecting the recombinant vector into a suitable host cell. Any host cells known in the pertinent art to enable stable and continuous cloning or expression of the recombinant vector may be used as the host cell. Suitable prokaryotic host cells may be one or more selected from E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus species strains such as Bacillus subtillis, or Bacillus thuringiensis, intestinal bacteria and strains such as Salmonella typhymurum, Serratia marcescens, and various Pseudomonas species. Suitable eukaryotic host cells to be transformed may be one or more selected from yeasts, such as Saccharomyces cerevisiae, insect cells, plant cells, and animal cells, for example, Sp2/0, Chinese hamster ovary (CHO) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines, but not be limited thereto.

The polynucleotide or the recombinant vector including the same may be transferred (transfected) into a host cell by using known transfer methods. Suitable transfer methods for prokaryotic host cells may include a method using CaCl₂ and electroporation. Suitable transfer methods for eukaryotic host cells may include microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene bombardment, but are not limited thereto.

In another aspect, provided is a method of preparing an anti-c-Met antibody with improved c-Met inhibition, anticancer effects, and/or angiogenesis inhibitory effects, or a method of improving c-Met inhibition and/or angiogenesis inhibitory effects of an anti-c-Met antibody, including coupling an anti-c-Met antibody or an antigen-binding fragment thereof (or a modified form of the antibody) with a VEGF-binding fragment as described herein. The coupling can be effected by any suitable technique, such as by synthesizing a nucleic acid encoding the c-Met antibody or fragment thereof and the VEGF-binding fragment as a fusion protein, optionally including a linker sequence between the c-Met antibody and the VEGF-binding fragment.

When an existing anti-c-Met antibody is coupled with a portion capable of binding to VEGF (for example., Ig-like domain 2: VIG2 (e.g., SEQ ID NO: 110) of human VEGF Receptor 1 (e.g., SEQ ID NO: 109) or a polypeptide comprising 102 to 1338 consecutive amino acids containing SEQ ID NO: 110 among SEQ ID NO: 109), to be manufactured as a bispecific (dual-targeting) antibody form that targets c-Met and VEGF at the same time, it is confirmed that the bispecific antibody exhibits synergistic effects while maintaining the effect of targeting each of c-Met and VEGF.

Accordingly, the manufacturing technology of the bispecific antibody proposed in this invention may improve the anticancer effects of anti-c-Met antibodies.

The thus prepared bispecific antibody can more efficiently inhibit the growth of cancer cells than the anti-c-Met antibody alone, and even when bispecific antibodies as described above were manufactured using various kinds of anti-c-Met antibodies, it was confirmed that they exhibited superior effects to the single antibodies (see Example 5). That is, the manufacturing method of the bispecific antibody proposed in this invention is generally applicable to enhance the anticancer effects of the anti-c-Met antibodies.

The bispecific antibodies maintained c-Met binding related properties including anticancer and anti-angiogenesis effects of the existing anti-c-Met antibodies (see Example 2). Also, the bispecific antibodies antagonized the biological functions of VEGF by including a VEGF-binding region (see Example 3). Furthermore, the anticancer effects of the bispecific antibodies were confirmed to be superior to the anti-c-Met antibodies through animal model-based tests (see Example 4).

Further, even in the cases of various forms of anti-c-Met antibodies (different linkers, different lengths, modified Fc portion, different CDR, etc.), they exhibited similar effects when coupled with VIG2 (see FIG. 4A).

In this respect, the method of attaching the VEGF-binding region to the anti-c-Met antibody is expected to be generally applicable to enhance the anticancer and anti-angiogenesis effects of anti-c-Met antibodies.

Also, as the fusion protein and/or the bispecific antibody enable simultaneous detection of c-Met and VEGF, they are useful in the simultaneous detection of c-Met and VEGF, and diagnosis of c-Met and VEGF induced diseases. An embodiment provides a method of detecting c-Met and VEGF or diagnosing a c-Met and VEGF induced disease, including contacting the fusion protein and/or the bispecific antibody to a sample, and measure the level of binding of the fusion protein and/or the bispecific antibody and both of c-Met and VEGF.

As the technology of coupling the anti-c-Met antibody with the VEGF-binding region provided in this invention enhances anticancer/angiogenesis inhibitory effects of the anti-c-Met antibody, it can be used for the development of anticancer drugs applicable to brain cancers, lung cancers, stomach cancers, etc., and drugs of diseases induced by angiogenesis mechanism. Additionally, this technology can be used for the development of drugs against diseases with which c-Met/HGF signal transduction system and VEGF/VEGFR signal transduction system are associated. Accordingly, excellent effects can be achieved, as well as simple administration using the bispecific antibody when compared to the co-administration of related drugs.

One or more embodiments of the present invention will now be described in further detail with reference to the following Examples. However, these examples are for the illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES Reference Example 1 Construction of Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1 Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the first injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tail and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of phosphate buffered solution (PBS). The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸ cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in water at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO₂ incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (orthphenylenediamine; OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, with Accession No. KCLRF-BP-00220 according to the Budapest Treaty (refer to Korean Patent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody was produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with a filter (Amicon). The antibody in PBS was stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mice antibody AbF46 produced in Reference Example 1.1 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, to construct a vector including the heavy chain and a vector including a light chain for the expression of the chimeric antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 μl (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

Thereafter, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, 100 mL of the supernatant was collected and applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (www.ncbi.nlm nih.gov/igblast/) result revealed that VH3-71 has a homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDRs of the mouse antibody AbF46 into the framework of VH3-71. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzed by a search for BLAST. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST (www.ncbi.nlm.nih.gov/igblast) search result revealed that VK4-1 has a homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest homology with the VL gene of the mouse antibody AbF46 were analyzed by a BLAST search (www.ncbi.nlm.nih.gov/igblast). As a result, VK2-40 was selected as well. VL and VK2-40 of the mouse antibody AbF46 were found to have a homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A BLAST search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Hereupon, back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy; SEQ ID NO: 47, H3-heavy; SEQ ID NO: 48, H4-heavy; SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50; H2-light: SEQ ID NO: 51; H3-light: SEQ ID NO: 52; H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (Invitrogen) (A), and in another 15 ml tube, 100 μl of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples comprised a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of scFV Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker having the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) coding for the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDR and Synthesis of Primer

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 1, below.

TABLE 1 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG (SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 2 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.

TABLE 2 Library con- Clone structed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides coding for heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment comprising L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment comprising L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment comprising L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment comprising L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (Invitrogen) (A), and in another 15 ml tube, 100 μl of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain comprising the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge and the constant region of human IgG1 constant region, and a light chain comprising the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain comprising a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain comprising the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain comprising the light variable region of huAbF46-H4-A1 and a human kappa constant region. Hereupon, the histidine residue at position 36 on the human kappa constant region of the light chain was changed into tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, (i) a polynucleotide (SEQ ID NO: 63) coding for a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, (ii) a polynucleotide (SEQ ID NO: 65) coding for a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, (iii) a polynucleotide (SEQ ID NO: 67) coding for a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and (iv) a polynucleotide (SEQ ID NO: 69) coding for a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 μl of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1(U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was representatively selected for the use in the following examples, and referred to as L3-1Y in the following examples.

Example 1 Preparation of Fusion Proteins

The anti-c-Met antibodies manufactured in Reference Example 1 were fused to a linker by coupling the C-terminal of their heavy chain therewith. Thereafter, an Ig2 domain, that is, amino acids from 129^(th) to 229^(th) among the amino acids constituting VEGF receptor 1 was sequentially fused at the terminal of the linker to manufacture antibodies capable of binding to c-Met and VEGF at the same time (see FIG. 1).

Among 1338 amino acids constituting VEGF receptor 1 (P17948.2; SEQ ID NO: 109), a gene sequence encoding 101 amino acids from 129^(th) to 229^(th) constituting the Ig2 domain which has been shown to be most important for VEGF-binding was secured from NCBI database.

Ig2 domain (VIG2) amino acid sequence (SEQ ID  NO: 110): SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFP LDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKT NYLTHRQTNTI Ig2 domain (VIG2) nucleotide sequence (SEQ ID  NO: 111): AGTGATACAGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCC GAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGC CGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCA CTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGT AGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGG CTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACA AACTATCTCACACATCGACAAACCAATACAATC

In order to couple the heavy chain of the c-Met antibody manufactured in the above and the Ig2 domain (VIG2), three types of linkers were designed having repeating GGGGS motifs: ‘GGGGS’(G4S), (SEQ ID NO: 116) ‘GGGGSGGGGS’((G4S)₂) (SEQ ID NO: 117), or ‘GGGGSGGGGSGGGGSGGGGS’((G4S)₄) (SEQ ID NO: 118). The peptide linker was to be placed between the c-Met antibody and the Ig2 domain (VIG2) of VEGF receptor 1. Accordingly, a gene was synthesized that encoded the heavy chain of the anti-c-Met antibody, the linker sequence, and the VEGF-binding fragment. The synthesized gene also included a stop codon (TGA) at the end of the designed final gene. The synthesized gene was inserted into pOptivec vector (Invitrogen) using an EcoRI/XhoI cloning site to produce a heavy chain expression vector. The vector used in the manufacture of L3-1Y was used as a light chain expression vector.

Each of the thus constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and the vector including the heavy chain and the vector containing the light chain were transfected at the ratio of 4:1 into 293T cells (2.5×10⁷ cells) to which 360 μl of 2 M CaCl₂ was added. Thereafter, the transfected cells were cultured in a DMEM medium containing 10% FBS at 37° C. in 5% CO₂ conditions for 5 hours, and then cultured in an FBS-free DMEM medium at 37° C. in 5% CO₂ conditions for 48 hours.

The cultured cells were centrifuged to obtain 100 ml of each supernatant, which was purified using AKTA Prime (GE Healthcare). Protein A column (GE Healthcare, 17-0405-03) was placed in the AKTA Prime, and the cultured solution flowed at a rate of 5 ml/min and was eluted with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with a PBS buffer, and thus antibodies capable of binding to cMet and VEGF at the same time were finally purified.

The thus prepared antibodies are summarized in Table 3:

TABLE 3 Heavy Constant Light VEGF-binding Name Chain Hinge Region Linker Chain fragment Single- L3-1 SEQ ID U6 HC7 (SEQ IgG1 — SEQ ID — targeted NO: 62 ID NO: 101) NO: 70 antibody L3-1Y SEQ ID U6 HC7 IgG1 — SEQ ID — NO: 62 (SEQ ID NO: NO: 68 101) Bispecific MV10A SEQ ID U6-HC7 IgG1 (G4S)2 SEQ ID VIG2 antibody NO: 62 (SEQ ID NO: NO: 70 (SEQ ID NO: 111) 101) MV10AY SEQ ID U6-HC7 IgG1 (G4S)2 SEQ ID VIG2 NO: 62 (SEQ ID NO: NO: 68 (SEQ ID NO: 111) 101) MV10AY SEQ ID U3 HC9 (SEQ IgG1 (G4S)2 SEQ ID VIG2 U3 NO: 64 ID NO: 102) NO: 68 (SEQ ID NO: 111) HC9/ IgG1 MV10AY SEQ ID U3 HC9 (SEQ IgG2 (G4S)2 SEQ ID VIG2 U3 NO: 66 ID NO: 102) NO: 68 (SEQ ID NO: 111) HC9/ IgG2

Example 2 c-Met Target and Degradation Activity of Fusion Proteins

2.1. Affinity Test

The binding kinetics of the double-targeted fusion proteins manufactured in the above were analyzed using Biacore T100 instrument (GE Healthcare). A human Fab binding agent (GE Healthcare) was immobilized on the surface of a CM5 chip (GE Healthcare, Cat. No. BR-1005-30) according to the manufacturer's manual. About 90120 RU of L3-1Y or MV10AY was captured and various concentrations of c-Met (R&D Systems, Cat. No. 358-MT/CF) were introduced to the captured antibodies by injection. Then, 10 mM Glycine-HCl (pH 1.5) solution was added thereto, whereby the surface was regenerated. In order to measure the binding kinetics, the data obtained from the above experiment was fitted using BIAevaluation software (GE Healthcare, Biacore T100 evaluation software).

The results obtained by using the BIAevaluation software are shown in Table 4 below.

TABLE 4 Sample R_(max) (RU) K_(D) (nM) k_(a) (1/Ms) k_(d) (1/s) L3-1Y 89.63 <0.01 7.7 × 10⁵ <1.2 × 10⁻⁵ MV10AY 89.59 <0.01 6.8 × 10⁵ <1.4 × 10⁻⁵ MV10AY U3 102.5 <0.01 7.2 × 10⁵ <6.9 × 10⁻⁵ HC9/IgG2

The affinity of MV10AY toward c-Met was similar to that of L3-1Y (K_(D)<0.01 nM), regardless of the backbone isotype (IgG1 or IgG2) of the anti-c-Met antibodies.

2.2. c-Met Degradation ELISA (MKN45)

A relative total amount of c-Met was used to evaluate the effects of an antibody by figuring out the increase/decrease of the total amount of c-Met using the fact that the degradation of c-Met occurs by the internalization of the antibody when it binds to c-Met. It has been already shown that binding of c-Met to HGF promotes the growth of cancer cells and in this regard, the above experiment was created by the idea that if the total amount of c-Met is reduced, the growth of cancer cells decreases.

The amount of c-Met was measured by a quantitative ELISA method, and the experiment was performed in an MKN45 stomach cancer cell line (JCRB0254; Health Science Research Resource Bank (HSRRB, Shinjuku, Japan)), using human total HGF R/c-Met ELISA kit (R&D systems). Cells (200,000 cells/ml) were mixed with 5 μg/ml of anti-c-Met antibodies, and cultured in a medium (RPMI with 10% Fetal Bovine Serum) for 24 hours, followed by ELISA experiments. Lastly, they were reacted with Super Aquablue (eBiosciences) and their colorimetric signals were measured as OD values at the wavelength of 450 nm. The value of a control group (media) treated with no anti-c-Met antibody was set to 100%, and values obtained after treatment with the anti-c-Met antibodies were calculated as relative values against the control group.

The obtained results are shown in FIG. 2A. As seen in FIG. 2A, MV10A and MV10AY, which are bispecific antibodies, had significantly improved c-Met degradation ability in comparison with L3-1 and L3-1Y each of which is the corresponding c-Met single-targeted antibody. That is, the c-Met degradation ability of MY10A (76%) was significantly improved in comparison with the degradation ability of its single-targeted antibody L3-1 (68%) (1-way ANOVA analysis result p<0.01). In the case of MV10AY, its degradation ability (72%) was also improved more than the degradation ability of its single-targeted antibody (66%) (1-way ANOVA analysis result p<0.05).

2.3. Akt Phosphorylation (Caki) (Agonism Test of Antibodies)

Safety and efficacy for therapeutic antibodies were evaluated by mechanism-based experiments. A phosphorylation degree of an AKT kinase was measured by the quantitative ELISA method so as to examine safety. Cellular activities controlled by AKT include cell proliferation, cell survival, cell size regulation, reactivity to soluble nutrients, intermediate metabolism, angiogenesis, tissue invasion, etc. All these processes represent the characteristics of cancer, numerous oncoproteins and tumor suppressors influence reciprocally to one another on the AKT route, and they perform to minutely regulate the cellular activities at a connection point of signal transduction and classic metabolic regulation. Accordingly, as the degree of a phosphorylated AKT which is an active form of AKT gets higher, cancer cells become more active. In this regard, this example investigated how much the antibodies could inhibit the phosphorylation of AKT, when compared to the treatment by the positive control antibody 5D5.

A site to be phosphorylated in AKT is Ser 473, and PathScan phospho-AKT1 (Ser473) chemiluminescent Sandwich ELISA kit from Cell signaling Co. was used. A Caki-1 kidney cancer cell line (HTB-46; American Type Culture Collection (ATCC), Manassas, Va.), which was cultured to 200,000 cells/ml one day before, was mixed with 5 μg/ml of antibodies in a serum-free DMEM medium (GIBCO, Invitrogen), and treated for 30 min., followed by experiments using an ELISA kit. The results were obtained by measuring with a machine of Perkins Elmer Co. When the phosphorylation degree of AKT was to be calculated, the phosphorylation degree of positive control 5D5 (American Type Culture Collection; ATCC, Manassas, Va.) was set to 100%. In comparison with this, the phosphorylation induction degrees of other anti-c-Met antibodies and bispecific antibodies were expressed.

The obtained results are shown in FIG. 2B. As seen in FIG. 2B, the bispecific antibodies (MV10A, MV10AY) inhibited the phosphorylation of AKT significantly more strongly than their corresponding c-Met single-targeted antibodies (L3-1, L3-1Y). That is, the AKT phosphorylation inhibitory ability of MV10A (79% compared to 5D5 control group) was significantly improved in comparison with the inhibitory ability (69%) of its single-targeted antibody L3-1 (1-way ANOVA analysis result p<0.01). In the case of MV10AY, its inhibitory ability (74%) was also improved more than the inhibitory ability (63%) of its single-targeted antibody L3-1Y (1-way ANOVA analysis result p<0.01).

2.4. MKN45 Growth Inhibition Test (Antibody Efficacy Test)

A human stomach cell line MKN45 (JCRB0254) was obtained from Health Science Research Resource Bank (HSRRB, Shinjuku, Japan). The cell line was cultured in an RPMI1640 medium (GIBCO, Cat. #11875-119) containing 10% (v/v) fetal bovine serum (FBS, GIBCO Cat. #16000-044) and 1% (v/v) penicillin/streptomycin (GIBCO, Cat. #15410-122). The cell line was cultured at 37° C. under a humid atmosphere containing 5% CO₂, and it was subcultured before confluence. The number of the cells was measured using CEDEX Analyzer (Roche Diagnostics). Luminescent assay by Celltiter Glo (CTG: Promega Co.) was employed to examine tumor cell proliferation according to in vitro antibody treatment.

The above assay was carried out according to the manufacturer's manual. In brief, the MKN45 cells within the RPMI1640 medium containing FBS 10% (v/v) were seeded at a concentration of 1×10⁴ cells per well onto a black 96-well plate (Corning Incorporated, Cat. #Costar 3603), which was then treated with the antibodies of which the final concentrations were diluted to 0.008 μg/mL, 0.04 μg/mL, 0.2 μg/mL, and 1 μg/mL using a 10% FBS containing RPMI 1640 medium. After incubation for 72 hours, 100 microliter of CTG solution was added to each well, followed by incubation at a room temperature for 30 min. The obtained luminescent signals were recorded using Envision 2104 Multi-label Reader (Perkin Elmer, Waltham, Mass., USA).

The obtained results are shown in FIG. 2C. As seen in FIG. 2C, bispecific antibody MV10A (▪) showed significantly excellent MKN45 cancer cell line growth inhibitory effects at 0.04 μg/mL or higher concentrations in comparison with its corresponding c-Met single-targeted antibody L3-1 (□) (average and standard deviation marked, 2-way ANOVA analysis; p values obtained from Bonferroni multiple comparison test are marked below each concentration point. **, p<0.01; ***, p<0.001; ****, p<0.0001).

2.5. NCI-H441 Growth Test (Agonism Test of Antibodies)

When c-Met single-targeted antibody binds to c-Met, undesired agonism phenomenon by c-Met occurs. Since this is associated with the safety of the antibody, a test to evaluate whether the agonism properties which the c-Met single-targeted antibody does not want are not increased by having double target specificity was carried out using an NCI-H441 cell line. The NCI-H441 cell line has a tendency of cell division by agonism under specific conditions described below when administered with c-Met single-targeted antibody.

The NCI-H441 cell line (ATCC Cat. #HTB-174) was diluted at 200,000 cells/ml in a serum-free RPMI1640 medium (Gibco). seeded at 100 μl/well onto a 96 well plate, and then cultured at 37° C., in 5% CO₂ conditions for 24 hours. After that, they were treated with anti-c-Met antibodies having 6 concentration gradients in 1/5 serial dilution starting from the concentration of 10 μg/ml. After the cells were cultured at 37° C., in 5% CO₂ conditions for 21 hours, 5-bromo-2′-deoxyuridine (BrdU) was added to be incorporated into the cells, which were further cultured for 3 hours, followed by BrdU assay (Roche, Indianapolis, Ind.). The cells were subjected to denaturation/fixation on a plate, anti-BrdU antibodies were added thereto, a substrate was added 1 hour thereafter, and a color reaction was measured using an ELISA spectraMax reader (Molecular Devices, Sunnyvale, Calif.). Lastly, after the subtraction of the value of cells into which BrdU was not incorporated as a background control, the value of a control group to which no anti-c-Met antibodies were treated was converted to 100% (Y axis) to calculate relative values. Mouse IgG was used as a negative control and a 5D5 antibody (isolated and purified from ATCC Cat. #HB11895 hybridoma cells) known to be an agonist was used as a positive control.

The obtained results are shown in FIG. 2D. As seen in FIG. 2D, the bispecific antibody MV10A or MV10AY did not increase agonism properties when compared to the c-Met single-targeted antibody L3-1. In other words, there were no changes in antibody safety.

2.6. HUVEC Proliferation Suppression Test (+HGF) (Anti-Angiogenesis Test)

A HuVEC cell line (ATCC) was cultured along with 0.4 μg/ml of HGF (R&D systems) in an EGM2 bulletkit medium (Lonza Cat. CC-3162). After the cells were incubated under a humid atmosphere containing 5% CO₂ at 37° C., they were subcultured before confluence. The number of the cells was measured using a CEDEX Analyzer (Roche Diagnostics). Real time cell analysis was conducted using Xcelligence Real time cell analyzer (Roche) in order to see tumor cell growth inhibition degree according to in vitro antibody treatment. This analysis was carried out according to the manufacturer's manual. In brief, the cultured HuVEC cells were seeded at a concentration of 1×10⁴ cells per well onto a 16-well E-plate, the incubated medium was removed after 24 hours, and the cells were treated with an antibody (100 μl, final antibody concentration 10 μg/mL) which was diluted in a free-serum medium (EBM media, Lonza Co.). The values of occurring impedances were measured at real time. Real time analysis was conducted for 48 hours.

The obtained results are shown in FIG. 2E. By virtue of these test results, it could be determined how much the single/bispecific antibodies could suppress the influence of HGF on the growth of HUVEC. As seen in FIG. 2E, Bevacizumab (Roche Korea, the same amount treated) did not have any influences on the effects of HGF, like the negative control (HGF only). Nonetheless, MV10A, the bispecific antibody of c-Met and VEGFR, showed stronger agonism effects on HGF than its original c-Met single-targeted antibody, L3-1 (MV10A 64% suppression, L3-1 45% suppression; compared to HGF only group, p value marked by 1-way ANOVA: *, p<0.05; NS, no significance).

Example 3 VEGF Target and Suppression Activity of Fusion Proteins

3.1. Affinity Test

The binding kinetics of the double-specific antibodies having VEGF as a target were measured by an SPR method using Biacore T100 instrument (GE Healthcare). hVEGF (GE Healthcare, 28-9583-25) was immobilized on the surface of a CM5 chip according to the manufacturer's manual. MV10AY having various concentrations (6 types of concentrations) of about 90 RU was captured. The obtained surface was regenerated by injecting 1M NaCl+30 mM NaOH at a fluid rate of 30 μl/min for 60 sec. In order to determine binding kinetics, the data obtained from the above experiment was fitted using BIAevaluation software (GE Healthcare) (K_(D=)=0.45 nM).

The obtained results are shown in Table 5.

TABLE 5 Sample R_(max) (RU) K_(D) (nM) k_(a) (1/Ms) k_(d) (1/s) MV10AY 92.30 0.45 2.4 × 0⁶ 1.1 × 0⁻³

3.2. HUVEC Proliferation Suppression Test (+VEGF)

Anti-angiogenesis effects were examined by investigating HUVEC proliferation degree according to in vitro antibody treatment using Xcelligence Real time cell analyzer (Roche). The analysis was carried out according to the manufacturer's manual. In brief, a HuVEC cell line (ATCC) was cultured along with 0.4 μg/ml of VEGF (R&D systems) in an EGM2 bulletkit medium (Lonza Cat. CC-3162). The cultured HuVEC cells were seeded at a concentration of 1×10⁴ cells per well onto a 16-well E-plate, the incubated medium was removed after 24 hours, and the cells were treated with the antibodies (100 μl, final concentration 10 μg/mL) which were diluted in a free-serum medium (EBM media, Lonza). The values of occurring impedances were measured at real time. Real time analysis was conducted for 96 hours.

The obtained results are shown in FIG. 3A. As seen in FIG. 3A, the antibody according to the invention and Bevacizumab (Roche Korea, the same amount treated) used as a positive control all showed VEGF-dependent cell proliferation, and MV10A showed significant cell proliferation suppression effects, compared to its positive control, Bevacizumab (MV10A 86% suppression, Bevacizumab 61% suppression; compared to VEGF only group, p value marked by 1-way ANOVA: *, p<0.05; NS, no significance).

3.3. HUVEC Proliferation Suppression Test (+VEGF+HGF)

Anti-angiogenesis effects were examined by investigating HUVEC proliferation degree according to in vitro antibody treatment using Xcelligence Real time cell analyzer (Roche). The analysis was carried out according to the manufacturer's manual. In brief, cells (HuVEC, ATCC, 10000 cells/well) were treated with 0.4 μg/ml of VEGF (R&D systems) which is a ligand of c-Met and VEGFR and cultured in an EGM2 bulletkit medium (Lonza Cat. CC-3162). The cultured HuVEC cells were seeded at a concentration of 1×10⁴ cells per well onto a 16-well E-plate, the incubated medium was removed after 24 hours, and the cells were treated with the antibodies (100 μl, final concentration 10 μg/mL) which were diluted in a free-serum medium (EBM media, Lonza). The values of occurring impedances were measured at real time. Real time analysis was conducted for 48 hours.

The obtained results are shown in FIG. 3B. As seen in FIG. 3B, while the single administration of the single-targeted (c-Met) antibody L3-1Y and Bevacizumab (VEGF single target) antibody did not result in any significant HuVEC cell proliferation suppression effects, the bispecific antibody MV10AY showed significant synergistic suppression effects against HGF- and VEGF-dependent HUVEC cell proliferation. In other words, while the sole use of Bevacizumab showed 27% suppression and the sole use of L3-1Y showed only 16% suppression, the double-targeted MV10A antibody showed 72% suppression effects. Thus, when the bispecific antibody was used, the significant synergistic suppression effects were observed, compared to when the single antibodies were used (significance marked by 1-way ANOVA test with Bonferonni multiple comparison test. compared to HGF+VEGF only group at the top of the bar graph p value marked (***, p<0.001; NS, no significance), differences between each treatment group marked at the top of the graph).

3.4. HUVEC Invasion Inhibition Test (+VEGF)

In order to see whether in vitro antibody treatment inhibits cell invasion, the cell invasion was tested using a plate for cell invasion assay (BioCoat Growth Factor Reduced MATRIGEL Invasion Chamber; BD science, Cat#. 354483) which is divided into an upper chamber and a lower chamber and the upper chamber of which is coated with collagen. The assay was carried out according to the manufacturer's manual. In brief, HUVEC cells (ATCC; 5×10⁴ cells/well) were seeded into the upper chamber, and the lower chamber was treated with the antibody (10 μg/ml), which was then cultured at 37° C. (VEGF added to the lower chamber, 0.4 μg/ml). After 24 hours, the cells which went down to the lower chamber by penetrating the collagen layer of the upper chamber were dyed with calcein and observed using a fluorescence spectroscopy (ZEISS).

The obtained results and the fluorescence images are shown in FIG. 3C. The upper portion of FIG. 3C is a graph showing the relative fluorescence image areas of each antibody treatment group. They are relative values against the invasion occurring when treated with VEGF only (100%). The lower portion of FIG. 3C contains photographs illustrating actual fluorescence images (white indicates the cells which caused invasion). As seen in FIG. 3C, the double-specific antibody MV10AY showed significant invasion suppression effects in comparison with Bevacizumab, the VEGF single-targeted antibody (MV10AY 100% suppression, Bevacizumab 70% suppression).

3.5. HUVEC Invasion Inhibition Test (+VEGF+HGF)

In order to see whether in vitro antibody treatment inhibits cell invasion, the cell invasion was tested using a plate for cell invasion ability assay (BioCoat Growth Factor Reduced MATRIGEL Invasion Chamber; BD science, Cat#. 354483) which is divided into an upper chamber and a lower chamber and the upper chamber of which is coated with collagen. The assay was carried out according to the manufacturer's manual.

In brief, HUVEC cells (ATCC; 5×10⁴ cells/well) were seeded into the upper chamber, and the lower chamber was treated with the antibody (10 μg/ml), which was then cultured at 37° C. (HGF+VEGF added to the lower chamber, 0.4 μg/ml each). After 24 hours, the cells which went down to the lower chamber by penetrating the collagen layer of the upper chamber were dyed with calcein and observed using a fluorescence spectroscopy (ZEISS).

The obtained results and the fluorescence images are shown in FIG. 3D. The upper portion of FIG. 3D is a graph showing the relative fluorescence image areas of each antibody treatment group. They are relative values against the invasion occurring when treated with VEGF+HGF (100%). As the values go lower, it means that less invasion occurs. The averages and standard deviations are marked, and p values with regard to VEGF+HGF treatment group as a result of 1-way ANOVA analysis are each marked at the top of the graph (*, p<0.05; ***, p<0.001). The lower portion of FIG. 3D contains photographs illustrating actual fluorescence images (black indicates the cells which caused invasion). As seen in FIG. 3D, the double-specific antibody MV10AY showed remarkably excellent inhibitory effects against HuVEC cell invasion in the presence of VEGF and HGF in comparison with L3-1Y antibody, the c-Met single-targeted antibody (MV10AY 92% suppression; L3-1Y 79% suppression; and Bevacizumab 83% suppression).

3.6. HUVEC Migration Inhibition Test (+VEGF+HGF)

Both c-Met and VEGF are factors associated with not only cell growth but also cancer metastasis. It was tested whether the bispecific antibody has synergistic effects on cell migration related to cancer cell metastatic potential. To evaluate metastatic potential inhibition according to in vitro antibody treatment, the following experiments were carried out. Since cell migration is directly involved in the metastasis of cancers, the following analysis is commonly used to evaluate cell metastatic potential.

Oris 96-well plate (Platypus, Oris™ Cell migration assay) method was performed. HuVEC cells (ATCC) were seeded at 10000 per well onto a 96-well plate equipped with a stopper and treated with 0.4 μg/ml of HGF and 0.4 μg/ml of VEGF in a serum-free medium (EBM, Lonza). Then, after culturing for 24 hours, the stoppers were removed. Because the stopper is a circular rubber material, which prevents cells from growing at a place where the stopper was once placed, the removal of the stopper after 24 hours would result in a circular space at only that place.

After the removal of the stoppers, the cells were treated with anti-c-Met antibody L3-1Y, anti-VEGF antibody Bevacizumab, and MV10AY antibody at various concentrations (0.05 to 5 μg/ml). Then, after 24 hours, they were dyed with calcein AM (BD) which is a fluorescent substance, so that only the cells were dyed and the portions with no cells remained as spaces. Hence, as cell migration is inhibited more, the size of the space which is not dyed will get bigger and in this regard, cell migration degree can be measured by observing the space which is not dyed. The strength of fluorescence signals was read by a multilabel reader (Perkin-Elmer, Envision), and the obtained values were converted on the basis of the signal strength (100%) of VEGF+HGF treatment group and shown in FIG. 3E. The average and standard deviation at each concentration are shown, and their statistic significances with regard to VEGF+HGF group were marked at the top of each concentration point (2-way ANOVA with Bonferonni multiple comparison test; * p<0.05; *** p<0.001; **** p<0.0001).

At the treatment amounts of the antibodies within all the tested ranges, the bispecific antibody MV10AY showed remarkably excellent inhibitory effects, compared to the treatment of the c-Met single-targeted antibody (L3-1Y) or the VEGF single-targeted antibody Bevacizumab or the co-treatment of these single-targeted antibodies (synergism observed). This result undoubtedly shows the superiority of the MV10AY antibody which is an anti-c-Met antibody with added anti-VEGF function.

Example 4 In Vivo Anticancer Effects of Fusion Proteins

In vivo anticancer effects were tested using a U87-MG brain cancer cell line known to have anticancer effects when c-Met/HGF and VEGF are each suppressed. 2.5×10⁶ (100 μl) of the U87-MG brain cancer cell line (purchased from cell bank of Chinese Academy of Sciences; ATCC catalog no. HTB-14) were subcutaneously injected into BALB/c Nude mice (males, 6-7 weeks, 20-25 grams). After 1 week passed, the mice were divided into two groups: treatment groups as illustrated in FIG. 4A (MV10AY (▪), MV10AY U3 HC9/IgG1 (□), or MV10AY U3 HC9/IgG2 (◯); administration amount: 0.2 mg/kg each (dotted line) or 1 mg/kg (solid line); administration time: every 3-4 days or 10 days; administration route: intravenous injection) and control group (vehicle: PBS 0.2 mL administered by intravenous injection once a week), followed by drug administration. There were 15 mice in each group. Tumor volumes were measured twice a week, and the tumor volumes were calculated as {long diameter (mm)×short diameter (mm)²}/2. After 30 days from the treatment, the mice were euthanized, the tumors were extracted therefrom, and the weights of the tumors were measured and then used for the evaluation of anticancer effects along with the tumor volumes measured in the above.

The obtained results are shown in FIG. 4A. The upper portion of FIG. 4A is a graph illustrating tumor volumes according to time, and the bottom portion of FIG. 4A is a graph illustrating the weights of the tumors that were extracted after the completion of the experiment. As seen in FIG. 4A, all the treatment groups showed remarkable tumor inhibitory effects after two weeks from the administration. Treatment effect differences among the three bispecific antibodies were not significant, and the groups in which the administration amounts and administration intervals were increased showed much stronger inhibitory effects. The groups in which 1 mg/kg was injected every 3-4 days showed almost perfect anticancer effects to such an extent that tumors were hardly observed (top graph repeated measures ANOVA analysis, ** (p<0.01), **** (p<0.0001); bottom graph 1-way ANOVA Dunnett's multiple comparison test analysis with regard to the Vehicle group, ** (p<0.01), *** (p<0.0001)).

In order to see whether the bispecific antibody that targets c-Met and VEGF at the same time shows more improved anticancer effects than the c-Met single-targeted antibody, an in vivo anticancer effect experiment was conducted using a NCI-H441 cell line. NCI-H441 is a cell line which shows anticancer effects as well as agonism effects by virtue of a c-Met target, and was determined to be the most suitable cell line to see comprehensive anticancer effects. 5×10⁶ (100 μl) of the NCI-H441 lung cancer cell line (purchased from ATCC, Cat. #HTB-174) were subcutaneously injected into BALB/c Nude mice (males, 5-6 weeks, 20-25 g). After 1 week passed, the mice were divided into two groups: treatment groups (single-targeted antibody L3-1Y and bispecific antibody 5 mg/kg each, administered once a week) and a control group (vehicle: PBS 0.2 mL administered by intravenous injection once a week), followed by drug administration. There were 15 mice in each group. Tumor volumes were measured twice a week, and the tumor volumes were calculated as {long diameter (mm)×short diameter (mm)²}/2. After 26 days from the start of the treatment, the mice were euthanized, the tumors were extracted therefrom, and the weights of the tumors were measured and then used for the evaluation of anticancer effects along with the tumor volumes measured in the above.

The obtained results are shown in FIG. 4B. The upper portion of FIG. 4B is a graph illustrating tumor volumes according to time, and the lower portion of FIG. 4B is a graph illustrating the weights of the tumors that were extracted after the completion of the experiment. As seen in FIG. 4B, statistically significant anticancer effects were observed after one week from the start of administration in both of the two treatment groups. Especially, when treated with MV10AY, stronger anticancer effects were observed than when treated with L3-1Y (top graph repeated measures ANOVA analysis, **** (p<0.0001); bottom graph 1-way ANOVA with Dunnett's multiple comparison test analysis vs the Vehicle group, * (p<0.05), *** (p<0.0001)).

Example 5 Fusion Proteins Comprising Various Anti-c-Met Antibodies

It was examined whether the efficacy of anti-c-Met antibodies having other CDRs (in addition to L3-1, L3-1Y, or L3-1Y IgG2 which were illustrated in the above examples) is improved when fused with VIG2.

The efficacy of the anti-c-Met antibodies was measured by testing c-Met degradation degree according to the same method as described in Example 2.2, and the antibodies used are summarized in Table 6 below.

TABLE 6 Name Heavy Chain Light Chain Linker VEGF-binding fragment Ab1 SEQ ID NO: SEQ ID NO: — — 112 113 Ab1- SEQ ID NO: SEQ ID NO: (G4S)₂ VIG2 VIG2 112 112 (SEQ ID NO: 110) Ab2 SEQ ID NO: SEQ ID NO: — — 114 115 Ab2- SEQ ID NO: SEQ ID NO: (G4S)₂ VIG2 VIG2 114 115 (SEQ ID NO: 110)

The obtained results are shown in FIG. 5. As seen in FIG. 5, the bispecific antibodies, Ab1-VIG2 and Ab2-VIG2 improved c-Met degradation ability compared to their c-Met single-targeted antibodies Ab1 and Ab2, respectively. That is, the c-Met degradation ability (83.2%) of Ab1-VIG2 was improved in comparison with the degradation ability (81.7%) of its single-targeted antibody Ab1. In the case of Ab2-VIG2, its degradation ability (81.9%) was improved more than the degradation ability (70.4%) of its single-targeted antibody Ab2 (t-test analysis result p<0.01).

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

1. A fusion protein comprising (a) an anti-c-Met antibody or an antigen-binding fragment of the anti-c-Met antibody and (b) a VEGF-binding fragment.
 2. The fusion protein according to claim 1, wherein the VEGF-binding fragment is an anti-VEGF antibody or antigen-binding fragment thereof, or a VEGF receptor or VEGF-binding region thereof.
 3. The fusion protein according to claim 2, wherein the VEGF-binding fragment is (i) bevacizumab, (ii) an antigen-binding fragment of bevacizumab, (iii) a VEGF receptor comprising SEQ ID NO: 109, (iv) a VIG2 polypeptide comprising SEQ ID NO: 110, or (v) a polypeptide comprising 102 to 1338 consecutive amino acids of SEQ ID NO: 109 comprising SEQ ID NO:
 110. 4. The fusion protein according to claim 1, further comprising a linker between (a) the anti-c-Met antibody or the antigen-binding fragment of the anti-c-Met antibody and (b) the VEGF-binding fragment.
 5. The fusion protein according to claim 4, wherein the linker is a peptide linker comprising 1 to 100 amino acids.
 6. The fusion protein according to claim 1, wherein the anti-c-Met antibody specifically binds to an epitope comprising 5 to 19 contiguous amino acids of SEQ ID NO: 71, and wherein the epitope comprises the amino acid sequence of SEQ ID NO:
 73. 7. The fusion protein according to claim 1, wherein the anti-c-Met antibody comprises: a heavy chain variable region comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid sequence comprising 8-19 consecutive amino acids of SEQ ID NO: 2 comprising the amino acid residues from the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 85, or an amino acid sequence comprising 6-13 consecutive amino acids of SEQ ID NO: 85 comprising the amino acid residues from the 1^(st) to 6^(th) positions of SEQ ID NO: 85; and a light chain variable region comprising at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 86, or an amino acid sequence comprising 9-17 consecutive amino acids of SEQ ID NO: 89 comprising the amino acid residues from the 1^(st) to 9^(th) positions of SEQ ID NO:
 89. 8. The fusion protein according to claim 7, wherein the CDR-H1 has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, the CDR-H2 has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 25, or SEQ ID NO: 26, the CDR-H3 has the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 85, the CDR-L1 has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 106, the CDR-L2 has the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, and the CDR-L3 has the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 86, or SEQ ID NO:
 89. 9. The fusion protein according to claim 7, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 74, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 75, SEQ ID NO: 88, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO:
 107. 10. The fusion protein according to claim 7, wherein the anti-c-Met antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 62, the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64, the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66, or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and a light chain comprising the amino acid sequence of SEQ ID NO: 68, the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70, the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, or the amino acid sequence of SEQ ID NO:
 108. 11. The fusion protein according to claim 1, wherein the anti-c-Met antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO: 114; and a light chain comprising the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO:
 115. 12. The fusion protein according to claim 1, wherein the anti-c-Met antibody is a monoclonal antibody.
 13. The fusion protein according to claim 1, wherein the anti-c-Met antibody is a mouse originated antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.
 14. The fusion protein according to claim 1, wherein the fusion protein comprises an antigen-binding fragment of an anti-c-Met antibody selected from the group consisting of scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂.
 15. A bispecific antibody against c-Met and VEGF, comprising the fusion protein of claim
 1. 16. A method of preventing or treating a disease induced by c-Met or VEGF, comprising administering a pharmaceutical amount of the bispecific antibody of claim 15 to a patient in need thereof, thereby preventing or treating a disease induced by c-Met or VEGF in the patient.
 17. The method according to claim 16, wherein the disease induced by c-Met or VEGF is a cancer, gestational diabetes, diabetic retinopathy, or macular degeneration.
 18. A method of preparing an anti-c-Met antibody having improved efficacy, comprising coupling an anti-c-Met antibody or an antigen-binding fragment of the antibody, with a VEGF-binding fragment.
 19. The method according to claim 18, wherein the VEGF-binding fragment is (i) a VIG2 polypeptide comprising SEQ ID NO: 110 or (ii) a polypeptide comprising 102 to 1338 consecutive amino acids of the amino acid sequence of SEQ ID NO: 109, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:
 110. 20. The method according to claim 18, wherein the VEGF-binding fragment is coupled to the anti-c-Met antibody or an antigen-binding fragment thereof through a linker.
 21. A nucleic acid encoding the fusion protein of claim 1, optionally in a vector.
 22. A method of preparing a fusion protein according to claim 1 comprising expressing a nucleic acid encoding the fusion protein in a cell. 